The primary goal of this research proposal is to characterize a steroid biotransformation in man and animals which is exclusively catalyzed by the host's intestinal bacteria. Recently, Eubacterium sp. number 144 and number 146 have been isolated and shown to convert 16 alpha-hydroxyprogesterone to 17 alpha-progesterone with the intermediate accumulation of delta16-progesterone. The biotransformation appears to be catalyzed by two separate enzymes, 16 alpha-hydroxyprogesterone dehydoxylase and delta16-progesterone reductase. Chemically similar steriod dehydroxylation-reductions catalyzed by other intestinal anaerobes are thought to be carried out by a single enzyme or enzyme-complex. Characterization of this steriod biotransformation will be achieved through the following specific aims: (1) purification and determination of the enzymatic properties of 16 alpha-hydroxyprogesterone dehydroxylase and delta16-progesterone reductase, (2) determination, by continuous culture, of the effects of growth rate and nutrient limitation on the ability of Eubacterium sp. number 144 to catalyze steroid biotransformation, and (3) determination of the structure and metabolism of a novel water-soluble delta16-progesterone derivative formed spontaneously in the presence of cysteine. This reaction represents a potential metabolic fate for 16 alpha-hydroxyprogesterone mediated by Eubacterium sp. number 144 not previously known.